Cloning protein of interest into lentiviral vector - help - I have no experience - (Jul/13/2019 )

Thank you so much. I wouldn?t have managed this far without your help! I will let you know how it goes and will probably need more help in the next stages!

Hi Bob

 

I was hoping you can help again.

 

I got my primers and did the PCR reaction. I ran 5ul of the 50ul PCR reaction on a 1% agarose gel and had a look at the products.

 

I am just not sure if I am right in thinking that my product size should be 2077 bp including the HA and GST tags? I have attached an image showing the PCR product separation on a powerpoint slide and have labelled what I think is the product. I am not familiar with DNA ladders - there are so many ladder bands, some which are not distinguishable at the top. So I marked the ladder going up from the bottom. Could you kindly check this for me. The DNA ladder I used is the Invitrogen 1 Kb Plus DNA Ladder: https://www.thermofisher.com/order/catalog/product/10787018 . I think my band is the brightest one as it correlates with what I think is the 2KB band?

 

 

Something I'm confused about - on the Addgene website, which shows the pRK5 plasmid I originally amplified my gene from https://www.addgene.org/19304/ , it says the plasmid backbone size is 4754 bases and the insert is 1426 bp which comes to a total of 6180 but if you click on 'view all sequences' under the plasmid map image and go into 'analyze sequence' under the header 'Full sequences from Addgene' then look at the total number of bp in the plasmid - it is 6685. I don't understand why there is this discrepancy? Furthermore, on my agarose gel image where I have ran the original pRK5 plasmid as a control before PCR, the band is showing up quite a bit higher than 7000 bases - I don't understand why that is. Could you please help me understand?

 

 

Finally, if I am correct in thinking the bright band is my PCR product of interest, to purify the PCR product, do I run the remaining 45ul of the 50ul PCR reaction on an agarose gel again and cut of my band of interest then clean up with a gel purification kit?

 

Thanks so so much!

Hi Natalia,

 

I have been following your thread and Bob1 has been helping you a lot.  I'll jump in- your PCRs are working and yes the strong band should be your product. If you want to produce cleaner PCR rxns, try putting much less template into the rxn. (Thanks for including the information on how much template you used on the attachment). A LOT less, like 0.1 ng or even down into the 50 picogram range. Dilute your template before adding. Your photo shows a lot of leftover plasmid and non-specific products that probably resulted from so much template. Remember the power of PCR is to amplify from tiny quantities to massive quantities, and when you are amplifying from plasmid, you only need a tiny bit to start. (Very different from amplifying from a eukaryotic genome.)  That way there is a negligible amount of plasmid left in your finished reaction.

 

That said, you can gel-purify your band from the remaining PCR rxn with a gel purification kit, and continue from there, as you said.  Or if you do a cleaner reaction with much less template, and you only see your desired product on test sample you run on the gel, you can purify the rest of the 50 ul rxn with a PCR clean up kit (make sure you use one for your size product) that uses a spin column to get rid of the leftover primers, nucleotides, etc. without gel purification.  The advantage to this is you do not expose your purified band to ethidium or UV light, so you don't get nicks, and you avoid the possibility of any agarose contamination in your product. And it is easier.  In any case run a sample of your purified product on a gel and see how pure it looks; don't just depend on a Nanodrop reading for quality.

 

Actually, looking at your gel, you have so much product that if you ran a whole 50 (or 45) ul on a gel it would make a big blob, and be hard to purify.  You have more than enough showing on your gel to isolate and subclone the band as it is. My experience has taught me that sometimes less is more.

 

It has been a while since this thread has been active, so let us know how you are doing, and if you need any more help.

Hi OldCloner,

 

thank you for your response. I ended up running the remaining PCR product, which was gigantic in size like you said it would be but I did a gel purification and sub cloned the PCR product into an entry vector (pDONR221) with an LR reaction. I then transformed this into bacteria and did mini preps for a few clones, after which I sent them for sequencing. I sequenced with M13 forward primer. 

 

I've never sequenced anything but I opened the files sent to me with 4peaks and guessed that it would be sufficient to copy and paste a section of the plasmid sequence into 4 peaks to see if it matched. Out of the 8 clones I mini prepped and sequenced, 2 of them matched a portion of my original insert. I then took these two clones and carried out a BP reaction to transfer my insert into gateway destination vectors (pLenti pCMV Blast and pLentiCMV Neo). Once again after transformation I did some mini preps and sent for sequencing. Most of the colonies I tested matched sections of my insert. I am now maxi prepping 1 or 2 for future use.

 

However, as I am using a universal primer, I only managed to sequence the HA and GST tags but not the remaining inset endowing my protein of interest. I wondered if you could advise what the best way of checking the remaining insert would be? I'm assuming as I managed too see the beginning of my insert, it is safe to assume that the whole insert is present in my plasmid?

 

Thanks for the help and advice!

Congratulations, it sounds like you have successfully got your cloning to work. 

 

The best way is to ensure that you have coverage from one end of your insert to another. This allows you to see if there were any errors in the sequence that might affect the open reading frame.

 

To do this, design some primers that are spaced about every 300-400 bases apart on the sequence. This should ensure that you cover the entire sequence with adequate overlap to rule out sequencing errors.  If you note a particular error in your sequence, design a reverse primer about 100 bp away from that site so that you can check if it is just a sequencing error or not. 

 

By the way - Check out Snapgene viewer for a good, free plasmid viewer. It is capable of taking sequences and doing all the plasmid mapping based on automated detection of sequence features etc. The full version is not free and comes with some really nice features that would be handy, but the free one covers most applications.

Like Bob1 I recommend that you sequence the entire insert; even the best proofreading polymerases can and do make errors, especially if there is anything funky about your insert (long single base repeats, or high or low CG content, etc.). In our lab it was considered a requirement to sequence anything cloned from PCR products. Bob gave you good advice on designing primers. If you become an "Old Cloner" you might stretch the intervals to 500-600 bases, but for now 300-400 is good. (I had the advantage of working in a sequencing lab, did my own sequencing and got really good at estimating how far my reads would go. I also ordered the internal primers, with the amplification primers, at the start of the project, to save time and shipping costs.)  It is good to sequence at least one clone before continuing to your final product; keep the other plasmids in case you do find an error, and you can check another one if your first one has an error. Also I would check to make sure you got the appropriate hybrid recombination sites for the Gateway reactions. Congrats on getting this far as a first time cloner!

Thanks! Let?s hope I get good data from these experiments!

Sorry I didn?t get what you meant by ?appropriate hybrid recombination sites for the Gateway reactions??

Is there any real difference between sequencing primers and other primers such as qPCR primers for example? Is there anything I should be aware of that needs to be done differently when designing sequencing primers?

Thanks again for all your help - was a great learning curve! My next project is to design crispr knock out guides and clone into appropriate lentiviral vectors so will start a new post on that in a few days! Any help on that from yourselves and anyone else would be great!

Thanks

What OldCloner meant was that there are sites on the plasmid - on either side of your insert - that are the sites that the gateway cloning system uses to get from one plasmid to another. These are usually designated AttR or AttB or something similar. If these sites are mutated, the gateway system will not work, so he was saying that you should confirm these sequences are correct too.

 

Primers are primers - the ones you use in real-time/qPCR are no different to those for regular PCR. If you are using a Taqman system then there are also the probes which are different and not the same as primers in that they insert between the primer sites. Basically however, they all boil down to the same thing - an oligonucleotide sequence that binds DNA. There are no special considerations for sequencing, just do the regular annealing temp/GC content things. You don't need these in pairs - only one primer per reaction for seqencing, as I am sure you already know. 

One small point regarding primers for sequencing. With Sanger sequencing, longer primers are recommended. For pcr, primers of ~20 bases are recommended. For sequencing, unless the recommendation has changed in the past 2 years, primers of ~30 bases are recommended. This makes them a little more specific and a little less prone to slippage.

That being said, I used to sequence successfully with pcr primers.

Well, here's a different opinion: I usually design sequencing primers of about 20 base pairs, and try to get a 50 to 60 percent GC content. Avoid repeat areas like "AGCAGCAGCAGC" and similar. Sanger sequencing usually involves an annealing temperature of 50 degrees C, so the Tm of your seq primers should be a bit higher.

 

It is true, however, that using longer primers for sequencing helps you get reads that begin closer to the primer.  You may have noticed that your chromatogram begins with some poor quality jumbles of colored peaks that you have to edit off.  With longer primers, even the shortest fragments generated in the PCR rxn are longer, so they are not as prone to getting lost in the ethanol precipitation step that precedes electrophoresis.(That is part of the reason the beginning of chromatograms are so poor)  But if you have a lot of overlap between your chromatograms, you usually don't have to worry about reading close to the primer. And yes, you can often sequence with a single primer originally designed for PCR. If you are not too familiar with how Sanger sequencing works there are some decent videos on YouTube.

 

Check your Gateway manual; it will explain how the recombination sites work and how you get a hybrid recombination site when it is done. The resulting att site is a little different than either of the two sites you started with. The manual should show the original sequences and the resulting hybrid. Manuals are great educators! If you don't have a paper copy, you can find it on-line.

 

The only difference between PCR primers and sequencing primers is that for PCR, you are often adding "tails" at the 5 prime end: tags, restriction sites and whatnot, so they wind up being quite long, sometimes.  Also you have to take into account, in designing them in pairs, that they don't stick to each other to make the dreaded "primer dimer."

 

As for qPCR, primer design can be a bit trickier, depending on your template and purpose. Not so much the length or nucleotide composition of the oligo itself, but where in the target sequence you put them. And what  protocol you are using for detection: Taq Man or Sybr Green are the most common methods. It is too complicated to get into now, but if you ever need to do qPCR you can ask for help here with your specific project. Or read manuals that come with the kits- often they help a lot.

 

Good luck!